Technical Abstract:
Soil is a source of primary inoculum of Aspergillus flavus and A. parasiticus, fungi that belong to section Flavi and produce highly carcinogenic aflatoxins in peanuts. Aflatoxigenic fungi invade developing peanut seeds during conditions of late-season drought stress and elevated soil temperatures, and the highest concentrations of aflatoxins are found in damaged seeds. Biological control of aflatoxin contamination in peanuts has been achieved by applying competitive, nontoxigenic strains of A. flavus and/or A. parasiticus strains to soil. In this study, a laboratory procedure was developed in which viable fungus-free peanut seeds are wounded and inoculated with field soil containing natural populations of fungi, then incubated under optimal conditions (0.92 seed water activity; 30 ºC) for invasion by section Flavi species. The procedure provided a model system for studying fungal competition, specifically the effect of soil density of (1) native Aspergillus species and (2) applied biocontrol fungi on the colonization of peanut seeds by aflatoxigenic Aspergillus species and associated aflatoxin formation. In an examination of 20 soils containing natural fungal populations, wounded peanut seeds were preferentially colonized by section Flavi species despite low soil densities of the section (2-1700 CFU/g) relative to the total numbers of filamentous fungi (' 1%). Colonization of seeds by individual section Flavi species (A. flavus, A. parasiticus, A. caelatus, A. tamarii and A. alliaceus) decreased as soil densities of other, potentially competing species within the section increased; a significant interactive effect (P < 0.0001) among soil densities in the colonization of seeds was detected. Soil densities of section Flavi species and A. niger showed a similar interactive effect (P < 0.0001). In a second series of experiments, conidia of two identifiable nontoxigenic biocontrol strains, A. parasiticus NRRL 21369 (orange-brown conidial mutant) and A. flavus NRRL 21882 niaD (nitrate-nonutilizing mutant), were separately added at increasing amounts to soil in which wild-type Aspergillus populations had been fully characterized according to composition, density, and toxigenicity. Aspergillus species colonizing peanut seeds were similarly characterized, and seeds were individually analyzed for aflatoxins. Aflatoxigenicity was high (> 98%) among wild-type strains of A. flavus and A. parasiticus from soils and on colonized seeds. Increasing soil densities of biocontrol strains resulted in decreasing incidences of aflatoxigenic A. flavus/A. parasiticus on seeds (R2 = 0.91 and 0.67 for NRRL 21369 and 21882, respectively; P ' 0.01) and in decreasing seed concentrations of aflatoxin B1 (R2 = 0.80 and 0.68; P ' 0.01). Increasing incidences of biocontrol strains on seeds similarly lowered incidences of wild-type A. flavus/A. parasiticus (R2 = 0.65 and 0.79; P < 0.05) and aflatoxin B1 (R2 = 0.69 and 0.90; P ' 0.01). Predictive mathematical relationships indicated that a 1:1 ratio of NRRL 21882 niaD to wild-type A. flavus/A. parasiticus would reduce aflatoxin B1 in seeds by 50% whereas only a 1.6:10 ratio is required with NRRL 21369 for the same effect. Reduction of aflatoxin contamination in peanuts by nontoxigenic strains is strain dependent, and the effectiveness of strains depends upon the composition and toxigenicity of the native Aspergillus populations and on the densities of those populations relative to densities of applied nontoxigenic strains.